Cell specific gene expression in the genesis and dispersal of Vibrio cholerae biofilm

Vibrio cholerae causes the waterborne diarrheal disease, cholera, with annual fatality rates reaching 120,000 worldwide. Cholera outbreaks are common after natural disasters including earthquakes, storms, and floods where a reservoir of V. cholerae persists as aquatic biofilms (surface-attached microbial communities that are composed of microorganisms and a matrix composed of extra-polymeric substances, such as exopolysaccharides, proteins, and nucleic acids) found in sewage contaminated waters. Biofilms are complex structures consisting of cells which have distinct functions. The goal of this project is to identify the cellular states occupied by different V. cholerae cell populations within biofilms. We will determine how different cell types organize in 3-dimentional space during biofilm structure formation and how specific cells bring about the dispersal of biofilm in response to host signals present at the onset of infection. We will first measure the single cell transcriptome of thousands of single V. cholerae cells grown in laboratory culture media in either planktonic state or a biofilm using a microfluidic bacterial single- cell RNAseq assay we recently developed. We will compare the level of cell-to-cell heterogeneity in biofilm vs planktonic state at different times of growth. This data will identify the physiological states in which bacteria exist within the biofilm. After examination of the inherent cell-cell heterogeneity within biofilms we will use a fluorescence microscopy approach to determine the spatial arrangement of each cell-type within the 3-dimentional structure of biofilms. During V. cholerae colonization of the human GI tract ingested biofilms disperse into single cells upon contact with host-secreted bile acids. After we characterize the early formation, organization and maturation of biofilms we will next determine which cellular changes occur during biofilm dispersal in a model that utilizes specific bile-acids and temperature for biofilm dispersal. Our data will provide the first transcriptome-wide measurements of the transcriptional plasticity of V. cholerae in biofilms, their natural infective reservoir, and will be useful in identifying states associated with the onset of infection.